To determine whether cellular proteins are effective targets for small molecule inhibitors of enveloped RNA virus entry, this project will define the cellular factors required for the entry of a large panel of viruses selected from the families Alphaviridae (Chikungunya, Eastern equine encephalitis), Arenaviridae (Lassa, Machupo, Lujo), Bunyawiridae (Rift Valley fever. La Crosse), Coronavlridae {SARS, human coronavirus Erasmus Medical Center), Filoviridae (Ebola, Marburg), and Rhabdovlridae (rabies, Australian bat lyssavirus, Bas Congo). These families include some ofthe most lethal emerging viruses, for which there are no drugs or vaccines. We will engineer the envelope proteins of each into the vesicular stomatitis virus (VSV) genome and rescue autonomously propagating recombinant viruses to screen against a haploid human cell line to identify host genes required for infection. This will identify both virus-unique hits and hits that are shared among different viruses, reflecting the shared biology of their entry pathways. Many of the recombinant VSV are now in hand, and we have conducted successful screens for Ebola and Lassa that almost exclusively identified hits in these viruses' entry pathways, including receptors and transport factors. Prioritizing viral receptors and host factors shared by multiple viruses, we will define the roles of interesting candidate entry genes using gene knockout cells obtained by cloning from pools of mutagenized cells, purchase from commercial sources, or generation using TALEN endonucleases. We will conduct biochemical and genetic studies to define the role of specific genes in entry, and cell biological studies (in conjunction with Project 4) to define the cellular compartments in which the host genes function in viral entry. We will supply the gene inactivated cells to collaborators to show that the authentic BSL3 and 4 pathogens depend on these genes for infection in cell culture. We will use the most promising genes to adapt existing high-throughput assays to screen for small molecules that block engagement of specific receptors or that target host genes shared for the entry of many viruses. Promising inhibitors will be target-validated and assessed for ability to block infection of authentic viruses; validated inhibitors and host targets will be fed into the pipeline established by Projects 1 and 2. Although ambitious, the success of this project will at minimum yield a rich biology of host genes involved in the entry and pathogenesis of multiple viruses, and a set of small molecule inhibitors. This approach also provides a platform for rapidly identifying host proteins co-opted by newly identified human pathogens, which will be instructive in considering approaches to combat those infections.